1. Field of the Invention
The present invention relates to a process for isolating a pharmaceutically-active extract from a plant root, and more generally, to a novel method for preparing a medicament containing this extract. More specifically, the present invention relates to a process for maximizing the yield of valerenic acid and other valerenic acids and their derivatives, while simultaneously minimizing the yield of valepotriates and valepotriates decomposition products or derivatives in an extract of the root of the plant Valeriana officinalis L.
2. Description of the Related Art
Extracts of the root of the plant Valeriana officinalis L. (V. officinalis L.) have been used for medicinal purposes for over a century. Certain valerian extracts, including aqueous extracts, are known to have sedative and anxiolytic effects, but the active components have not been clearly and positively identified. [Leathwood P. D., Chauffard F., Heck E., and Munoz-Box R. Aqueous extract of valerian root (Valeriana officinalis L.) improves sleep quality in man. Pharmacology, Biochemistry and Behavior, 17:65-71, 1982; Leathwood P. D. and Chauffard F. Aqueous extract of valerian reduces latency to fall asleep in man. Planta Medica, 2:144-148 (1985)] Such effects are described by Balandrin et al. in U.S. Pat. No. 5,506,268, which is incorporated by reference herein in its entirety. Presently, valerian extracts are available as dietary supplements; these dietary supplements primarily comprise dried root or extracts from the root, formula into tablets or soft gelatin capsules. Each dose contains between approximately 50 mg and approximately 1 gram of dried root or extract. The use of these dietary supplements is extensive, with an estimated 210 million doses sold annually in the United States and 125 million doses sold annually in Europe. See Grunwald, J., xe2x80x9cThe European Phytomedicines Marketxe2x80x94Figures, Trends Analysesxe2x80x9d Herbal Gram, 1995.
V. officinalis L. is a member of the Valerianaceae family. This plant grows from a short rhizome to approximately 2 meters in height, it flowers, and then dies back again in the winter. Valeriana officinalis L. has pinnately-divided leaves, typically with six to ten pairs of lance-shaped leaflets, and bears many small white or pink flowers in a dense head of several stalked clusters. These heads bare small (5 millimeters) tapered seeds.
It is not fully understood which constituents of Valeriana officinalis L., and/or of the other heretofore unidentified members of the Valerianaceae family, are responsible for the sedative and/or anxiolytic action of valerian extracts. Nonetheless, the valepotriates (iridoids) as well as valerenic acid, a sesquiterpenoid compound, and the derivatives of valerenic acid (for example, acetoxyvalerenic acid and hydroxyvalerenic acid) along with the kessane derivatives, valeranone, valerenal, and certain amino acids are present in valerian extracts. Of these components, the valepotriates and valerenic acids are generally considered to contribute to the sedative action of valerian extracts, but have not been clearly and positively identified as such. See Hendriks H. et al. xe2x80x9cPharmacological Screening of valerenal and some other components of essential oil of Valeriana officinalisxe2x80x9d Planta Medica 42, 62-68 (1981); Bos R. et al., xe2x80x9cAnalytical aspects of phytotherapeutic valerian.xe2x80x9d (1996); Houghton P. J., Valerian. The Genus Valeriana. Harwood Academic Publishers, London (1997).
Valepotriates (iridoids) are triesters of polyalcohols with an iridoid structure. They are unstable, thermolabile compounds that decompose in alcoholic solutions and under acidic or alkaline conditions. See Bos R. et al. Analytical aspects of Phytotherapeutic Valerian preparation, 1996; 7:143-151. The major decomposition products of diene type valepotriates are baldrinals, including: baldrinal (from the valepotriates valtrate and acevaltrate) and homobaldrinal (from the valepotriate isovaltrate). Relative to the above-described valepotriates, valerenic acid and its derivatives (for example, acetoxyvalerenic acid and hydroxyvalerenic acid) are chemically stable. While valepotriates and valepotriate decomposition products or derivatives are found in many species of Valeriananceae, Valeriana officinalis L. is the only species that has been identified as possessing extractable valerenic acid in its roots. The structures of valerenic acids and certain valepotriates and valepotriate decomposition products are presented below. 
Several processes are known for the extraction of valerian roots, but none of these known processes simultaneously retains valerenic acid, maximizes the content of the valerenic acids (e.g., of valerenic acid and of the valerenic acid derivatives), and minimizes the content of valepotriates. Indeed, certain of these extraction processes were designed to retain or maximize the yield of valepotriates. For example, Broese et al., CH 000046778 (1978), describe obtaining a stable extract of a valerian preparation in which the essential valepotriates are present undiminished in the resultant extract, and Wischniewski et al., U.S. Pat. No. 4,313,930 (1982), describe the production of a stable valepotriate composition, through the use a pharmaceutically acceptable sheathing material, from a pharmaceutically active Valerianacea extract. Other known extraction processes focus on removing the odors associated with the valepotriate and valepotriate derivatives, but do not maximize the content of valerenic acid and the valerenic acid derivatives.
For example, Cerise et al., U.S. Pat. No. 5,211,948 (1993), describe extracting Valerian roots with water over a period of 2 to 5 hours at a temperature from 65 to 75xc2x0 C. This water-based extraction is described as leading xe2x80x9cto degradation of the valepotriates while preserving the valerenic acids of the valerian roots.xe2x80x9d The ""948 patent also states that extraction of valerian roots with hot water (2 to 5 hrs at 65-75xc2x0 C.), preferably three successive times, leads to degradation of the valepotriates while xe2x80x9cpreservingxe2x80x9d the valerenic acids. However, the results of the current examples, see esp. Examples 6-10, indicate that the valerenic acids are xe2x80x9cpreservedxe2x80x9d in the roots and are not extracted.
Valerenic acid is a chemically stable compound that is used as an identification test for V. officinalis in the United States Pharmacopoeia (USP23-NF18, Supplement 8). Thus, to develop a valerian based sedative product, it would be beneficial to enrich valerenic acids in the extract or substantially retain valerenic acid from the root. Due to poor solubility of valerenic acids in water, the process of the ""948 Patent is inefficient in extracting these compounds. The extraction process of the present invention, on the other hand, efficiently enriches valerenic acids. In this respect, the present invention is far more superior than the Cerise process.
Other extraction processes are known, but are conducted in the presence of unusual, expensive, or otherwise undesirable solvents and/or at unusual or undesirable conditions, such as low pH. For example, Thies et al., U.S. Pat. No. 3,422,090 (1969) describe extracting roots and rhizomes of plants of the genus Valeriana at a temperature below 30xc2x0 C. with a lipophilic solvent in the presence of an aliphatic carboxylic acid, specifically glacial acidic acid, within a pH range of about 3 to about 7. Gehrlicher, DE 03112737 (Mar. 31, 1981) (xe2x80x9cthe ""737 applicationxe2x80x9d) describes obtaining epoxide-free sedative agents from plants of the Valerianaceae family by extracting at elevated temperatures in the presence of weak acids and catalytic amounts of strong acids at pH less than 3, with either lower aliphatic alcohols or mixtures of lower aliphatic alcohols with water or anhydrous non-polar solvents. The process of the ""737 application, however, retains baldrinals and other valepotriate decomposition products in the extract.
A process for preparing a pharmaceutically-active extract of the root of a plant of the family Valerianacae, specifically, Valeriana officinalis L., is described. This process comprises the steps of adding the roots to an alcoholic extraction solvent to form a mixture, wherein the alcoholic extraction solvent comprises between approximately 50% to approximately 100% (v/v) ethanol in a remainder of water, and heating the mixture to a temperature of between approximately 70xc2x0 C. to approximately 80xc2x0 C. for a period of at least two hours. By this process the extract contains valerenic acids and has a content of valepotriates and valepotriate degradation products that is substantially reduced with respect to the content of valepotriates in the roots, and may have a content of valerenic acids that is not substantially reduced with respect to the content of valerenic acids in the roots, and also may have a content of volatile oils that is not substantially reduced with respect to the content of volatile oils in the roots. The process may be performed at pH greater than about 3.0, greater than about 5.0, or greater than about 7.0.
A pharmaceutically-active extract of the roots of a plant of the family Valerianaceae is also described. This extract is obtained by a process comprising the steps of adding the roots to an alcoholic extraction solvent to form a mixture, wherein the alcoholic extraction solvent comprises between approximately 50% (v/v) to approximately 100% (v/v) ethanol in a remainder of water, and heating the mixture to a temperature of between approximately 70xc2x0 C. to approximately 80xc2x0 C. for a period of at least two hours. This extract may then be used in a pharmaceutically active formulation preferably exhibiting sedative and/or muscle relaxant, and/or anxiolytic activity.
One objective of the present invention is to provide a process by which valerenic acid and its derivatives (e.g., acetoxyvalerenic acid and hydroxyvalerenic acid) are isolated in an extract of the roots of plants of the Valerianaceae family. More specifically, plants of the species Valeriana officinalis L. are used. Another, objective of the present invention is to provide a process by which valepotriates (iridoids) are substantially reduced in an extract with respect to their content in the roots, and preferably are not purified and/or isolated in an extract of the above-described roots. Another objective of the present invention is to provide a relatively inexpensive process that can be performed at commercial scale for the production of an extract having valerenic acids specifically, and valerenic acid and its derivatives (for example, acetoxyvalerenic acid and hydroxyvalerenic acid) generally, from the above-described roots. Another object of the present invention is to provide an extract process through which the extracted roots may be chemically identified as the roots of Valeriana officinalis L. Yet another object of the present invention is to provide a process for preparing a medicament containing an extract of the above-described roots. In the preferred embodiment of the extraction process of the present invention, but not necessarily in all embodiments of the extraction process of the present invention, these objectives are simultaneously met. Most preferably, the extract process of the present invention significantly reduces the amount of valepotriates in the extract and the amount of valerenic acid and/or valerenic acids is unaltered in the extract when compared to known processes.
According to the present invention, at least one of these objectives is met, at least in part, through an extraction process. In conjunction with this extraction process, methods for processing the root prior to extraction, for forming a drug substance via the addition of excipient, for drying and milling the drug substance, and for formulating an ingestible entity, such as a tablet, capsule, tea, suspension, or other medicinal food, are described. These methods provide a medicament of the present invention, which contains the extract of the present invention.
Shown below are structures of exemplary compounds isolated according to the preferred methods described herein. 
wherein, for the following compounds, R and Rxe2x80x2 are defined as follows:
Extracts of V. officinalis L. roots exhibit sedative and anxiolytic action. Pharmacology studies indicate that V. officinalis exerts sedative properties apparently by a combination of central nervous system (CNS) depression and smooth muscle relaxation. V. officinalis has been shown to decrease mobility, potentiate barbiturate induced sleep time, alter performance on rotorod and traction tests, act as an anti-convulsant and exert anti-spasmolytic properties. Studies in animals (mainly rodents) show that individual constituents of V. officinalis exert similar sedative and pharmacological properties, although they are unable to account for the total activity of the plant See Houghton P. J., Valerian. The Genus Valeriana. Harwood Academic Publishers, London (1997).
Studies intended to attribute the activity of plant extracts to individual constituents typically take into account the amount of one or more active compound(s) present in the plant extract and the concentration which shows activity in a test system. HPLC analysis of the extract obtained from the current process shows that the extract may contain approximately 0.0xe2x80x94to approximately 0.50% and preferably 0.30% valerenic acid, approximately 0.0 to approximately 0.50% and preferably 0.37% acetoxyvalerenic acid, and approximately 0.0 to approximately 0.1% and preferably 0.01% hydroxyvalerenic acid. Analysis of the extract by steam distillation shows that the extract may contain approximately 0.0 to approximately 5% of total volatile oils and preferably 1%, 2%, 3%, or 4% of total volatile oils.
Valerenic Acids
Valerenic acids are components of V. officinalis extracts, and of extracts of other members of the family Valerianaceae, that are likely responsible for the sedative activity of valerian. The valerenic acids (for example, valerenic acid, acetoxyvalerenic acid and hydroxyvalerenic acid) of V. officinalis may exert sedative properties. For example, valerenic acid administered intraperitoneally (IP) at a dose of 100 mg/kg, caused a decrease in motor activity, a decrease in respiratory rate, absence of righting reflex, ataxia, a decrease of abdominal muscle tone and a decrease in rotarod performance in mice. See Hendrick H, Bos R, Allersma D, Malingre T, and Koster A. Pharmacological Screening of Valerenal and some other Components of Essential Oil of Valeriana officinalis. Planta Medica: 1981; 42: 62-68. In a follow up study, the same researchers showed that valerenic acid influenced performance on both the rotarod and traction tests with statistical significance obtained at 100 mg/kg, IP. Valerenic acid was also shown to potentiate pentobarbital induced sleeping time at doses of 50 and 100 mg/kg, IP. See Hendricks H, Bos R, Woerdenbag H and Koster A, Central Nervous Depressant Activity of Valerenic Acid in the Mouse. Planta Medica: 1985; 51: 28-31. The anti-convulsant properties of valerenic acid have been characterized, see Hiller K. and Zetler G. xe2x80x9cNeuropharmacological studies on ethanol extracts of Valeriana officinalis L.: Behavioral and anticonvulsant propertiesxe2x80x9d Phytotherapy Res. 10 (1996) 145-151; it was shown that valerenic acid antagonized picrotoxin induced convulsions at 12.5 and 25 mg/kg, IP in mice. At a concentration of 1 mmole/liter, valerenic acid and acetoxyvalerenic acid were shown to inhibit the enzyme system which catalyses breakdown of GABA in the brain by 20% and 38%, respectively. See Riedel E, Hansel R and Ehrke G. Inhibition of GABA catabolism by valerenic acid derivatives. Planta Medica: 1982 ; 46: 219-220. An increase in brain concentrations of GABA, a potent central nervous system (CNS) depressant, may thus lead to a decrease in CNS activity and sedation.
One aspect of the current method is useful in verifying that the particular roots to be extracted are roots of the species V. officinalis L. and not of another plant, or more specifically of another member of the Valerianaceae family. And because many of the plants in this family are phenotypically similar and are therefore capable of being substituted for one another, a preferable means to identify V. officinalis L. is to identify valerenic acid directly in the extract.
Preferred extraction processes according to the invention, unlike known methods of extracting V. officinalis L. roots from water, yields a measurable amount of valerenic acid. As shown herein, see esp. Examples 6, 7, 8, 9, and 10, the extraction of V. officinalis L. roots in water yields substantially less valerenic acids than the methods described herein. As noted above, the present extraction method is therefore preferable to, and provides benefits not obtainable through the use of known water-based extraction methods. For example, the present process allows for the extraction of a pharmaceutically-effective composition that can be independently verified as being from the roots of V. officinalis L., rather than from the roots of another plant or of a phenotypically-similar member of the Valerianaceae family.
In another embodiment of the invention, the amount of total valerenic acids or of a specific valerenic acid may be a marker for the strength/potency of the extract. For example, 0.6%-1.0% total valerenic acids may serve as such a marker.
Volatile Oils
Another preferred aspect of the extracts and extraction methods provided herein that is distinct from known valerian extractions and extraction methods concerns the yield of a measurable amount of volatile oil in the extract. As shown in, for example, Example 10, described in detail below, the extraction of V. officinalis L. roots in water does not yield isolatable volatile oil.
The volatile oils of V. officinalis are very complex mixture of mostly mono- and sesquiterpenoids. Over 150 compounds have been identified in the volatile oil fraction and have been described. See Bos R. Anaylytical and Phytochemical Studies on Valerian and Valerian Based Preparations. Thesis: Offsetdrukkerij Ridderprint B. V., Ridderkerk: 1997; 13-21 and 77-93.
Several studies have reported sedative effects of the total volatile oil and/or volatile oil constituents (kessoglycol diacetate, kessoglycol 2-acetate, kessylglycol 8-acetate, valerenal, and valerenone) in in vivo animal models of sedation. See Hazelhoff B, Malingre T and Meijer D. Antispasmodic effects of valeriana compounds: an in-vivo and in-vitro study on guinea-pig ileum. Arch Int Pharmacodyn Ther.: 1982; 257(2): 274-87; Hendricks, et al. 1981; Hikino H, Hikino Y, Kobinata H, Aizawa A, Konno C and Oh""Izumi Y. Sedative Principles of Valeriana Roots. Shoyakugaku Zasshi: 1980; 34(1): 19-24. Thus, the volatile oil may contribute to the overall sedative properties of the extract.
However, even the most abundant volatile oil is generally a minor component in the extract. Thus, individual components of the volatile oil-will not typically, but may, be justified as a useful marker for monitoring the consistency of the extract across manufactured batches.
In another embodiment of the invention, therefore, the amount of total volatile oils may be a marker for the strength/potency of the extract. For example, approximately 0.6% to approximately 2.2% total volatile oil may serve as such a marker.
The volatile oil content typically ranges from approximately 0% to approximately 5.0%, and preferably approximately 0.6 to approximately 2.2% (v/w).
In sum, the pharmacology studies cited in the literature indicate that volatile oil components of V. officinalis may be related to sedative activity. The current process may retain this component and the assay of the total volatile oil component, as the measurement from steam distillation indicates that the product has about 0.0% to about 5% of volatile oil. The current method thus may provide a way of control for the total amount of volatile oil within the extract. Accordingly, the present extraction method is preferable to, and provides benefits not obtainable through, the use of known water-based extraction methods.
Valepotriates and Valepotriate Decomposition Products
Another aspect of the current method that is distinct from known valerian extraction method concerns the in-process reduction of the content of valepotriates in the extracts. Valepotriates are triesters of polyalcohols with an iridoid structure and an epoxy group that have reported cytotoxic and mutagenic properties. Valepotriates are divided into two main groups: diene type (which include valtrate, isovaltrate, and acevaltrate) and monoene type (which include didrovaltrate and isovaleroxyhydroxydidrovaltrate (IVHD). Valepotriates are thermolabile and decompose under acidic, alkaline or alcoholic conditions. See Bos, 1996. Shown below are chemical structures of certain valepotriates.
While valepotriates may contribute to the pharmaceutical activity of valerian extracts, valepotriates and their decomposition products are also potentially dangerous. For example, valepotriates have been shown to be cytotoxic in vitro, and exhibit potential mutagenic activity. See Von der Hude W., Scheutwinkel-Reich M., Braun R., and Dittmar W. xe2x80x9cIn vitro mutagenicity of valepotriatesxe2x80x9d Arch Toxicol 56 (1985) 267-71; Von der Hude W., Scheutwinkel-Reich M., and Braun R. xe2x80x9cBacterial mutagenicity of the tranquilizing constituents of Valerianaceae roots.xe2x80x9d Mutat Res 169 (1986) 23-7). Also, orally-administered valepotriates reach the brain and other organs in vivo, and have been shown to be capable of irreversibly alkylating DNA and proteins (Wagner, H. and Jurcic, K, Planta Med. (1980) 38:366-376). As cytotoxic and mutagenic effects have been reported for the valepotriates the present invention provides a method of control for the levels of this class of compounds. This process comprises adding the ground roots to an alcoholic extraction solvent and heating to a temperature between 70xc2x0 C. and 80xc2x0 C. for a period of at least three hours. By this process the extract contains valerenic acids but has levels of valepotriates that are substantially reduced compared to the content of valepotriates that occur naturally in the roots of the plant.
Valepotriates are not unique to the genus officinalis, but are known constituents of most Valeriana species. V. officinalis mainly contains valepotriates of the diene type (valtrate, isovaltrate, and some acevaltrate), whereas both V. wallichii and V. edulis also contain considerable amounts of the monoene type (didrovaltrate and isovaleroxyhydroxydidrovaltrate). See Houghton, 1997. A few studies explored the content of different valepotriates in V. officinalis root. The studies indicate that only the diene valepotriates, valtrate and isovaltrate, are present in significant amounts, and all other valepotriates are present in trace amounts (see Table 1, below).
The data presented in Example 11 confirms the above information.
In addition, according to a preferred aspect of the invention, a lower limit for total valepotriates in the extract may be set as a process condition. For example, valtrate and isovaltrate may be quantitatively monitored because they are present in significant quantities in the V. officinalis biomass. Total valepotriate levels in the extract produced by the current process should preferably be  less than 0.1%. This ensures that the current extraction process consistently reduces valepotriates in the extract regardless of the starting concentration in the original biomass.
Major decomposition products of the valepotriates are the baldrinals, including baldrinal (from valtrate and acevaltrate) and homobaldrinal (from isovaltrate). See Bos, 1996; Houghton, 1997. As described in Bos (1997), the baldrinals are chemically reactive and may subsequently form polymers, although no evidence of polymerization was provided. Chemical structures of exemplary baldrinals are provided below.
Genotoxicity has been reported for both baldrinal and homobaldrinal. The compounds showed direct mutagenic effects in vitro in the AMES assay and the SOS-chromo-test (von der Hude, et al. 1986). In addition, metabolic degradation of 14C-methacetin was distinctly inhibited in vivo by baldrinal (26 mg/kg) and homobaldrinal (31 mg/kg) administered either IP or PO to mice (Braun et al., Studies on the effects of Baldrinals on hemopoietic cells in vitro, 1986; 52: 446-450), indicating decreased liver function.
Accordingly, it is desirable to eliminate, or substantially reduce the level of, valepotriates and valepotriate decomposition products, such as baldrinals, in valerian extracts with respect to the levels of valepotriates in the root or biomass. The extraction method of the present invention achieves this objective, while simultaneously achieving the objective of extracting a measurable quantity of valerenic acid as described above.
Definitions
As used herein, the term xe2x80x9cvalerianxe2x80x9d refers to any plant of the Valerianaceae family possessing extractable valerenic acid in its roots, and therefore refers, at least to, the plant designated Valeriana officinalis L. or alternatively herein, V. officinalis L. This species includes all recognized subspecies of Valeriana officinalis L. Some of these subspecies are also commonly referred to, in alternative taxonomic systems, as: Valeriana exaltata J. C. Mikan, Valeriana nitida Kreyer, Valeriana palustris Wibel, Valeriana wolgenis Kazak, Valeriana grossheimii Vorosch, Valeriana collina Wallr, Valeriana Rossica P. A. Smirn, Valeriana spryngini P.S. Smirn, Valeriana angustifolia Tausch, Valeriana tenuifolia Vahl, Valeriana wallrothii Kreyer, Valeriana ucrainica Demjan, Valeriana sambucifolia J. C. Mikan, Valeriana excelsa Poir, and Valeriana officinalis L.subsp. excelsa (Poir.) Rouy. Plants of the species Valeriana officinalis L. may be characterized as follows: These plant grows from a short rhizome to 2 m high, flowers, and then dies back again in the winter. These plant has pinnately-divided leaves with six to ten pairs of lance-shaped leaflets, and bears many small white or pink flowers in a dense head of several stalked clusters. These heads bare small (5 mm) tapered seeds.
As used herein, the term xe2x80x9cvalerian extractsxe2x80x9d most generally refers to the composition isolated from the roots of plants of the Valerianaceae family according to a specified extraction procedure, and preferably refers to the composition isolated from the roots of valerian or Valeriana officinalis L. according to a specified extraction procedure. These extracts comprise essential oils, valerenic acids, valepotriates (iridoids), kessane derivatives, valeranone, valerenal, fatty acids, carbohydrates and certain amino acids.
As used herein, the term xe2x80x9cvalerenic acidsxe2x80x9d refers to all chemically stable derivatives of valerenic acid. In its most limited, and preferable, connotation, this term refers to valerenic acid, acetoxyvalerenic acid, and hydroxyvalerenic acid (these three compounds, in aggregate, are also referred to herein as xe2x80x9cVAasxe2x80x9d). These compounds, either individually, in the aggregate, or based on their respective ratios, may be used as standards to evaluate the extract processes herein described and/or to evaluate the plant from which the extracted roots have been obtained. Valerenic acid, in particular, may be used to verify that the roots extracted are of the species Valeriana officinalis L.
Valerenic acid (also referred to herein as xe2x80x9cVAxe2x80x9d) is represented by the formula C15H22O2, has a molecular weight of 234.33 amu, a UV xcexmax at 218 nm with a log xcex5 of 4.232, and an [xcex1]20D of xe2x88x92117.8xc2x0 (c=1.64, EtOH). Acetoxyvalerenic acid (also referred to herein as xe2x80x9cAVAxe2x80x9d) is represented by the formula C17H24O4, has a molecular weight of 292.35 amu, a UV xcexmax at 217 nm with a log xcex5 of 4.184, and has an [xcex1]20D of 36.7xc2x0 (c=1.15, EtOH). Hydroxyvalerenic acid (also referred to herein as xe2x80x9cHVAxe2x80x9d) is represented by the formula C15H22O3, has a molecular weight of 250.34 amu, a UV xcexmax at 212 nm with a log xcex5 of 4.305, and has an [xcex1]20D of xe2x88x9298.4xc2x0 (c=0.63, EtOH). Accordingly, UV measurements at 220 nm may be used to determine the content of the valerenic acids in a given sample or aliquot.
As used herein, the term xe2x80x9cvalepotriatesxe2x80x9d (these compounds, in aggregate, are also referred to herein as xe2x80x9cVPsxe2x80x9d) refers to all chemically unstable, thermolabile triesters of polyalcohols having an iridoid structure that may be found in the roots of members of the Valerianaceae family. The most typical valepotriates, as that term is used herein, are the diene-type valepotriates, valtrate, acevaltrate, and isovaltrate. The decomposition products of valepotriates, for example, baldrinal and homobaldrinal are not within the definition of xe2x80x9cvalepotriates,xe2x80x9d but may be referred to as xe2x80x9cvaleportriate derivativesxe2x80x9d or xe2x80x9cvalepotriate decomposition products.xe2x80x9d UV measurements at 200, 254, and 320 nm are preferably used to determine the content of valepotriates in a given sample or aliquot.
As used herein, the term xe2x80x9cvolatile oilsxe2x80x9d refers to all oils in V. officinalis that are volatile, for example valerenal, valerenol, camphene, bornyl derivatives, myrtenyl acetate, and kessane derivatives.
As used herein, the term xe2x80x9cisolatedxe2x80x9d refers to the state of being free of other, dissimilar compounds with which the extracted components of the invention will normally be associated in their natural state, so that upon being xe2x80x9cisolatedxe2x80x9d the pharmaceutically-active components comprises at least about 0.25%, about 0.5%, about 1%, about 2%, about 4%, about 5%, about 10%, about 20%, about 50%, and at least about 75% of the mass, by weight, of a given sample.
As used herein, the term xe2x80x9cwaterxe2x80x9d refers to water, and preferably to potable water, which term includes purified and/or de-ionized water (DIW). Water, as used herein, may have dissolved within it a significant amount of any water-soluble solute, such as a salt or a sugar.
As used herein, the term xe2x80x9calcoholicxe2x80x9d refers to a process of or an extract obtained from extraction in an alcoholic extraction solvent containing a significant percentage of alcohol. xe2x80x9cAlcoholic extraction solventxe2x80x9d refers to extraction solvent having greater than approximately 10% alcohol by volume, and preferably refers to extraction solvents having at least approximately 25%, 30%, 35%, 40%, 45%, or preferably 50% alcohol by volume. Most preferably, xe2x80x9calcoholic extraction solventxe2x80x9d refers to an extraction solvent having alcohol content equal to or greater than approximately 70% by volume, and includes solvents that are 100% alcohol. Preferable, xe2x80x9calcoholic extraction solventxe2x80x9d refers to any C1-C6 alcohol, for example, methanol, ethanol, butanol or propanol, or any combination thereof, and most preferably refers to denatured ethanol (approximately 95% ethanol and approximately 5% methanol) The term xe2x80x9cethanolxe2x80x9d should be understood as referring to denatured alcohol unless specifically identified otherwise.
As used herein, the term xe2x80x9crootsxe2x80x9d refers to all of subterranean portion of a specifically or generically identified plant, including, but not limited to, the roots, the rhizomes, and the stolons of the specifically or generically identified plant. Where the term xe2x80x9crootsxe2x80x9d is not modified by a specifically or generically identified plant, it will be understood that the term refers to the roots of the species, and sub-species of, Valeriana officinalis L.
Extraction Processes
The extraction processes provided herein most generally involve heating a mixture of the roots and an alcoholic extraction solvent for an extended period of time to obtain valerenic acid and valerenic acid derivatives in the extract, and to significantly reduce the amount of valepotriates in the extract. These processes, when compared to currently known processes, significantly reduce the amount of valepotriates in the valerian extract, while maximizing the amount of valerenic acid and of valerenic acid derivatives. It is contemplated that the extract, isolated according to the method of the present invention, may ultimately be used for a pharmaceutically active formulation.
The method includes an extraction process. An overall method for preparing such a pharmaceutically active formulation is described to place the extraction process in the context of the preparation of the pharmaceutically active formulation. The following five steps comprise a preferred method for preparing such a formulation: Pre-Extraction Processing of the Root, Extraction, Drying and Milling of the Drug Substance, and Formulation of a Tablet or Capsule. Additional or alternative steps, as well as the use of different pharmaceutical formulations, may be added without departing from this process.
Pre-Extract Processing of the Root
The roots may be prepared for extraction by grinding, chipping, or pulverizing to a powder in a hammermill, or like an instrument, as will be appreciated by those of skill in the art. After such pre-extraction processing, preferably at least 70%, 75%, or 80%, and most preferably 85% or 90% of the mass of the roots pass through a Tyler 20-mesh screen. Also preferably, the raw or processed roots are stored in a durable non-reactive, preferably plastic, and more preferably polyethylene, container or containers. These containers may be doubly-lined with bags of like material and closed or closeable with a lid composed of like material.
Extraction
The valerian root, whether, as preferred, processed as described above or in an unprocessed state, may be added to an extraction solvent. Most preferably, the root is added in a ratio of approximately one kilogram to approximately five liters of extraction solution. The extraction solvent preferably is an alcoholic extraction solution, comprising between approximately 30% to approximately 100% (volume/volume; v/v) alcohol and between approximately 70% (volume/volume; v/v) to 0% (v/v) water. Preferably, the alcoholic extraction solvent comprises approximately 50% to approximately 100% (v/v), approximately 55% to approximately 95% (v/v), approximately 65% to approximately 85% (v/v), and approximately 65% to approximately 75% (v/v) alcohol. Specifically, the alcoholic extraction solvent may comprise approximately 50% (v/v), approximately 60% (v/v), approximately 70% (v/v), approximately 80% (v/v), approximately 90% (v/v) alcohol and approximately 100% alcohol. The alcohol used in the alcoholic extraction solvent is fully miscible in water, and is preferably denatured ethanol (95% ethanol+5% methanol), but may be any C1-C6 alcohol, including but not limited to methanol, ethanol, n-butanol, isobutanol, n-propyl alcohol, and isopropyl alcohol.
The mixture of root and alcoholic extraction solvent may be stirred by any mechanical device conventionally known for such purpose, including but not limited to an overhead stirrer, a magnetic stirrer assembly, and/or a built-in stirrer, and may be suitable for or adapted to the particular extraction vessel employed. The mixture may be heated to between approximately 65xc2x0 C. and 85xc2x0 C., and more preferably between approximately 70xc2x0 C. and 80xc2x0 C., or alternatively, the temperature of reflux. Specifically, the mixture may be heated to 50xc2x0, 55xc2x0, 60xc2x0, 65xc2x0, 70xc2x0, 75xc2x0, 77xc2x0, or 80xc2x0 or reflux. Various conventional methods may be used to heat the mixture, including but not limited to heating mantels or other resistive heating coils.
Preferably, the mixture is heated to any of the above-described temperature for at least one, one and one-half, two, two and one-half, three, three and one-half hours, four, or up to five hours. These durations, most preferably the latter three durations, are selected to significantly reduce the level of valepotriates relative to the initial value, preferably at least a 50% reduction. (Final Value/Initial Value=Percent Reduction). More preferably the reduction is by 60%, 70%, 75%, 80%, 90%, 95%, and most preferably 100% of the detectable level of valepotriates. In the latter case, the valepotriate level is not detectable by conventional techniques. The final valepotriate level may be obtained and may also be compared to that found in commercial valerian extracts.
Optionally, the mixture may then be cooled, preferably to room temperature or alternatively to a temperature above room temperature, including 30xc2x0 C., 35xc2x0 C., 40xc2x0 C., 45xc2x0 C., and 50xc2x0 C. The solids may then be separated from the liquid (by filtration or centrifugation or any other conventional method for separation) . The extraction vessel and the separated solids may be rinsed with the extraction solvent, described above. For such a rinse, from approximately four liters, three liters, two liters, or preferably one liter of extraction solvent may be used for each kilogram of root initially extracted.
Also optionally, the filtrate containing the extracted material may be concentrated to an oily consistency under reduced pressure, including approximately 0.9, 0.8, 0.7, 0.6, and 0.5 atmospheres (atms), at a temperature above room temperature, including 30xc2x0 C., 35xc2x0 C., 40xc2x0 C., 45xc2x0 C., and 50xc2x0 C. Optimally, a final volume of approximately 0.15 liters for each kilogram of root extracted is obtained.
Addition of Excipient to Facilitate Drying
The concentrate may be mixed with an excipient to facilitate drying. The excipient may be chosen from any commercially-available excipient or mixtures thereof, but is preferably selected from the following: maltodextrin-NF, tricalcium phosphate, silicon dioxide, dicalcium phosphate, microcrystalline cellulose, silicified microcrystalline cellulose, various ion exchange resins as will be understood by those of skill in the art, PVP/citrate, sodium citrate, pre-gelatinized corn starch (also known as Starch 1500), polyethylene glycol (PEG), sugar/polyol (also known as mannitol), TRIS buffer, sodium bicarbonate, porous silica, and combinations of the above-listed excipients and/or buffers, or other conventional excipient and any combination or mixture thereof as will be recognized by those of skill in the art. After addition of the excipient, the excipient will preferably comprise between approximately 10% and 40%, and more preferably between 20% and 25%, of the drug substance.
Drying and Milling of the Drug Substance
The concentrated valerian extract and excipient, if added, is dried under reduced pressure, including approximately 0.9, 0.8, 0.7, 0.6, and 0.5 atms, at a temperature slightly above room temperature, including 30xc2x0C., 35xc2x0 C., 40xc2x0 C., 45xc2x0 C., and 50xc2x0 C. Optimally, the drying is continuous until water content is equal to or less than less than 15%, 10%, or 5%, as measured by Karl Fischer analysis. The dried mixture may then be milled to a target of 80%, 85%, 90%, or 95% by weight passing through a size-exclusion screen of 60-mesh, 70-mesh, 80-mesh, 90-mesh, or 100-mesh.
Optionally drying of the extract may be accomplished by spray drying or any other conventional drying method as will be understood by one of ordinary skill in the art.
Certain of the constituents of V. officinalis L have been identified as sesquiterpenes (in the volatile oils) and iridoids (known as valepotriates). The total content of volatile oil varies widely within a single species, and also may vary between different species. The oil typically consists of mixtures of monoterpene and sesquiterpene derivatives. The amount of valepotriates also varies. The process of the present invention has been shown to reduce the amount of valepotriates when compared to currently practiced valerian extraction processes.
When verifying the plant source and process of the extraction process of the present invention, valerenic acid, acetoxyvalerenic acid, and hydroxyvalerenic acid are preferably used as marker compounds for analysis of the extract and later formulations based on the extracts. The process of the present invention significantly reduces the amount of valepotriates, while optimizing the yield of valerenic acid, acetoxyvalerenic acid, and hydroxyvalerenic acid either alone or in the aggregate. Active constituents of the extract of the present invention include, but are not limited to, valerenic acid and its derivatives (for example, acetoxyvalerenic acid and hydroxyvalerenic acid), kessane derivatives, valeranone, valerenal, small chain carboxylic acids, fatty acids and amino acids; the extract may also contains sugars and trace amounts of other aliphatic acids, alkaloids, phenolic acids, flavonoids, free fatty acids, sugars, and salts.
The present invention is herein described in detail through a variety of examples. It will be understood by those skilled in the art that the invention is not limited to the specific examples provided herein. Furthermore, although various amounts of plant material, specifically, V. officinalis L roots, and various other parameters under which extractions are performed, including specific pH conditions, temperatures, durations, and extraction solvents, are specified in the following examples, it will be understood by those skilled in the art that the invention is not limited to these specific plants, and these specific amounts and/or parameters. It will also be understood by those skilled in the art that the amount or type of plant material, the pH, temperature, solvent, and/or duration of extraction may be varied, and that the resultant process will still achieve one or more of the objectives of the invention.